RecQL4 tethering on the pre-replicative complex induces unscheduled origin activation and replication stress in human cells

Abstract

Edited by Patrick Sung Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.

A predictable conserved DNA base composition signature defines human core DNA replication origins

“…This result may suggest that the folded form of G4 participates in the initiation of DNA synthesis but is not required for origin recognition by pre-RC proteins. In agreement, MTBP, RecqL and Rif1, three factors involved in origin firing, all bind to G4 [49][50][51][52][53] .…”

“…This is also reminiscent of previous findings that origin density/origin activity is highly correlated with replication timing 3,25 . In addition, replication timing boundaries correlate with TAD boundaries 49 . Hence, altered DNA initiation density, aberrant replication timing and altered chromosomal structure organisation might be linked in cell types undergoing immortalisation.…”

Protective Mechanisms Against DNA Replication Stress in the Nervous System

“…The RECQL4 protein participates in HR and NHEJ DSB repair [155,156] and telomere maintenance [157]. Its roles in origin activation during the S-phase and chromosome alignment during replication were also described [158][159][160][161]. Different mice models of Recql4 inactivation have been generated, but no CNS phenotypes were found [162][163][164].…”

Congenital Diseases of DNA Replication: Clinical Phenotypes and Molecular Mechanisms

“…Sld3/Sld7 are needed to stabilize CDC45 binding to MCM2-7 until the GINS complex is recruited with Sld3, Sld2, Dpb11 and pol ε following S-CDK phosphorylation of Sld3 and Sld2 [ 19 , 28 , 29 ]. Similarly, in humans, TopBP1 and MTBP interact with Treslin in a CDK-dependent manner and are required for CDC45 recruitment and stabilization [ 23 , 24 , 30 , 31 ]. Unlike the role that Sld2 plays in GINS recruitment in yeast, RECQL4 has not been shown to interact with GINS but is required for replication initiation, possibly by stabilizing the CMG complex after GINS loading [ 22 , 31 , 32 ].…”

“…Similarly, in humans, TopBP1 and MTBP interact with Treslin in a CDK-dependent manner and are required for CDC45 recruitment and stabilization [ 23 , 24 , 30 , 31 ]. Unlike the role that Sld2 plays in GINS recruitment in yeast, RECQL4 has not been shown to interact with GINS but is required for replication initiation, possibly by stabilizing the CMG complex after GINS loading [ 22 , 31 , 32 ]. Furthermore, whereas Sld3, Sld7, Sld2 and Dpb11 are thought to dissociate from the replication fork after DNA melting, in humans, TopBP1 continues to travel with the elongating replication fork ( Figure 1 ) [ 33 ].…”